To compare the activity of RGD-TRAIL in different expression systems, RGD-TRAIL in both Escherichia coli (E.coli) and Pichia pastoris was constructed and expressed. In vitro activity of RGD-TRAIL from Pichia pastoris expression system was also analyzed. Genetic engineering techniques were used to construct recombinant plasmid pET30-rgd-trail and pHBM-rgd-trail. The recombinant protein RGD-TRAIL was purified with Ni ion affinity chromatography after induction. MTT assay, ELISA, scratch wound healing, transwell migration assay and Hoechst 33342 staining were performed to detect the effects of RGD-TRAIL on proliferation, binding activity, migration and apoptosis. The expression of apoptosis-associated proteins was detected by Western blotting. Recombinant protein RGD-TRAIL was successfully expressed in a form of inclusion body in E.coli, while expressed secretorily in Pichia pastoris. It possessed more potent cytotoxicity than RGD-TRAIL in E.coli by MTT assay. The RGD-TRAIL expressed by Pichia pastoris showed powerful binding affinity with cancer cells expressing α(v), DR4, DR5 and highly potent cytotoxicity through inducing apoptosis of cancer cells. Nuclear fragmentation was examined by Hoechst 33342 staining. Cleaved PARP and caspase-3 were also detected after incubation with RGD-TRAIL. Additionally, RGD-TRAIL inhibited migration significantly in A549 and HT1080 cells. The results demonstrate that Pichia pastoris expression system is more suitable for the recombinant protein RGD-TRAIL. Its binding affinity and antitumor activity might make RGD-TRAIL a promising candidate for cancer therapy.
Antineoplastic Agents ; pharmacology ; Apoptosis ; Blotting, Western ; Cell Line, Tumor ; Chromatography, Affinity ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Humans ; Oligopeptides ; biosynthesis ; pharmacology ; Pichia ; metabolism ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; biosynthesis ; pharmacology

Objective To analyze the common respiratory tract infection pathogens distribution and their drug resistance in pedi-atric cardiac intensive care unit(CICU),so as to provide reference for clinical rational use of antibiotics.Methods 1 350 cases of sputum specimens from lower respiratory tract infection patients of pediatric CICU in the medical center between January 2011 and December 2012 were cultivated and drug susceptibilities were tested.The results were retrospectively analyzed.Results 490 patho-genic strains were isolated from 1 350 cases of sputum specimens and identified,including Gram negative bacilli 288 strains (58.78%),Gram positive coccus 140 strains(28.57%),fungi 62 strains(12.65%,mainly Candida albicans ).Gram negative bacilli was given priority to with Klebsiella pneumoniae (62 strains,12.65%),followed by Branhamella catarhalis ,Pseudomonas aerugi-nosa and Escherichia coli .The rates of extended-spectrum beta-lactamases(ESBLs)-producing strains among Escherichia coli and Klebsiella pneumoniae were 73.33% and 66.13%,respectively.Gram positive coccus was given priority to with Staphylococcus aureus (65 strains,13.27%),followed by Streptococcus pneumoniae .Methicillin-resistant Staphylococcus aureus (MRSA)accounted for 24.62%.Conclusion Staphylococcus aureus ,Streptococcus pneumoniae and Klebsiella pneumoniae are main pathogens of re-spiratory tract infection in pediatric CICU.And there is multiple drug-resistant bacteria infection.Rational applicattion of antibiot-ics according to the test results of isolation and drug susceptibility is an effective way to control the infection of critical children and reduce the emergence of resistant strains.

Simplified interspecies nuclear transfer in mouse and bovine and the possibilities of the embryos reprogramming were developed. The zona and nucleus of the bovine oocyte were removed by pronase and bisection method respectively. When the skin fibroblast of mouse was fused with the nucleus-free oocyte of the bovine using 40?s and 1. 5 kv/cm direct current pulse, the rates of fusion and cleavage were 67. 44% and 30.23% respectively. Reconstructed heterogenous embryos finally developed to 8-cell stage when they were previously activated by ionomycin and 6-DMAP and subsequently cultured in press-made micro-well in the drop of the medium. Zona-free bisection method could be used for mouse heterogenous embryos cloning, and the micro well culture method could be used to avoid zona-free embryos aggregation.

Objective To compare the clinical efficacy of percutaneous kyphoplasty (PKP) by balloon dilatation between unipedicular and bilateral pedicle approach for the treatment of osteoporotic vertebral compression fractures (OVCF). Methods From January 2012 to March 2014, 56 patients with single-level or double- level OVCF were randomly divided into two groups. 29 patients with 33 vertebral bodies were performed by PKP through unipedicular approach and 27 patients with 31 vertebral bodies through bipedicular approach , used PMMA bone cement filling vertebral. The clinical data of patients were prospectively analyzed and the clinical efficacy was compared between two groups. Results The VAS scores, Beck value, Cobb angle and ODI scores after operation improved in both groups (P < 0.01),but there were no significant difference between two groups(P > 0.05). The operative time, X-ray exposure time and volume of bone cement in unilateral group were less than that of bilateral group(P < 0.01). There were no significant difference in the incidence of bone cement leakage ,the pain relief and the incidence of complications between two groups (P > 0.05). Conclusion Both unipedicular and biopedicular PKP are significant improvement for the OVCF, vertebral strengthen showed similar efficacy, but the former has easy operation procedure, can shorten operative time and reduce X-ray exposure time and less volume of bone cement.

Objective:To prepare and purify siRNA targeting a proliferation-inducing ligand targeted(APRIL-siRNA),so as to provxde a basis for studying the role of APRIL in human pancreatic cancer.Methods:pET-22b-APRIL was constructed to express APRIL dsRNA of human pancreatic cancer cell line CFPAC-1 in E.coli and the product was purified by chromatography using CF-11 column.APRIL dsRNA was digested by RNaseⅢto prepare APRIL siRNA,then the reaction mixture was loaded onto a DEAE ion exchange chromatography to remove RNaseⅢfrom oligonucleotides,and size exclusion chromatography was used to purify 21 bp siRNA.The purified APRIL siRNA was used to transfect Chinese hamster ovary(CHO)cells and the expression of APRIL in CHO cells was observed under fluorescence microscope Results:APRIL dsRNA was successfully expressed in E.coli after IPTG induction and was purified by CF-11 column.dsRNA was hydrolyzed with RNaseⅢand was purified by DEAE ion exchange chromatography and size exclusion chromatography.15% nondenaturing PAGE and 12% SDS- PAGE confirmed that RNaseⅢwas removed from oligonucleotides and 21 bp siRNA was purified with size exclusion chromatography.It was also found that APRIL siRNA obviously depressed APRIL expression in CHO cells.Conclusion:We have successfully constructed APRIL siRNA targeting APRIL gene of CFPAC-1 cells with in vitro transcription,which provides a basis for knock-down of APRIL gene in CFPAC-1 cells.

Objective To delineate the speculation that triple combination of small doses of calcium antagonist (levoamlodipine),the ACEI(imidapril)and the ARB(candesartan)may exert more significantly effect on mi- croalbuminuria(MAU)in patients with type 2 diabetes mellitus and hypertension.Methods One hundred sixty ca- ses of consecutive patients with type 2 diabetes mellitus and hypertension were randomized to receive following vari- ous combinations:amlodipine 2.5 mg/candesartan 4 mg(n=40),candesartan 4 mg/imidapril 5 mg(n=40);imidapril 5 mg/amlodipine 2.5 mg(n=40),and amlodipine 2.5 mg/candesartan 4,mg plus imidapril 5 mg(n=40).The changes in blood pressure and MAU were determined before and after treatment.Results Four combinations of treatment all significantly lowered BP(P

Isolated alkaloids from Coptis chinensis Franch. The compounds were identified as berberine, columbamine, groenlandicine, jatrorrhizine, magnoflorine, corydaldine and ferulic acid methylester. Then measured their bitter degree based on the electronic tongue and evaluated the antibacterial. The results based on the Electronic Tongue showed that berberine, columbamine, groenlandicine and jatrorrhizine have higher bitter degree than magnoflorine and corydaldine. And they also appeared better antibacterial activity on E. coli and S. aureus. The correlation coefficients between bitter degree and the two bacteria antibacterial activity were 0.983 and 0.911. So there was close relationship between the bitter degree and antibacterial activity of bitter components. Thus, it is confirmed further that bitter components are the material foundation of medicinal effectiveness of bitter herbs.
Aporphines ; analysis ; Berberine ; analogs & derivatives ; analysis ; Berberine Alkaloids ; analysis ; Biomedical Research ; instrumentation ; methods ; Coptis ; chemistry ; Drugs, Chinese Herbal ; analysis ; chemistry ; pharmacology ; Electronics ; instrumentation ; methods ; Escherichia coli ; drug effects ; growth & development ; Microbial Sensitivity Tests ; Reproducibility of Results ; Staphylococcus aureus ; drug effects ; growth & development ; Taste

Objective To observe the safety and efficacy of the posterior spinal instrumentation (PSI) with preserving of the intraspinal rib head in neurologically intact patients with scoliosis secondary to Neurofibromatosis type 1 (NF1).Methods The clinical and radiographic data of nine NF1 scoliosis patients with rib head protrusion into the spinal canal,who had undergone PSI from August 1998 to March 2012,were retrospectively investigated.The average age of these patients (4 males,5 females) was 15.4±7.6 years,and their neurological status all were Frankel grade E preoperatively.The intraspinal rib head was not resected in all patients intraoperatively.The following parameters,including the magnitude of rib head penetration into the spinal canal (MRPC),the angle between the bilateral rib (ABR),the angle between the dislocated rib and the posterior vertebral wall (ARV),the coronal Cobb angle and the sagittal Cobb angle,were measured before and after surgery.Results The follow-up period was 0.5 to 4.8 years.The average MRPC decreased from preoperative 32.8％±9.9％ to postoperative 16.8％±15.2％ (P=0.026); the average ARV increased from preoperative 34.7°±16.4° to postoperative 47.8°±17.5° (P=0.001); the average ABR decreased from preoperative 83.0°±19.5° to postoperative 67.9°±13.3° (P=0.009); the average coronal Cobb angle decreased from preoperative 70.7° ±17.7° to 35.4°±17.0° immediately after operation (P=0.000) and the sagittal Cobb angle decreased from preoperative 59.7°±17.6° to 24.7°±10.8° immediately after operation (P=0.001).The coronal Cobb angle and sagittal Cobb angle had no significant change during follow-up period.The neurological status was Frankel grade E in all patients immediately after operation and at final follow-up.Conclusion For NF1 scoliosis patients with rib head penetration into the spinal canal without impingement of the spinal cord and neurological deficits,the deformity can be corrected safely and effectively without resecting the intraspinal rib head.

Objective To investigate the value of lower limbs fixation methods for the treatment of intussusception in children with air enema with.Methods 2 1 1 pediatric patients with intussusception who had accepted the treatment of air enema with two dif-ferent fixation methods on lower limbs were enrolled.Comparisons of median treatment duration and therapeutic effect between the two methods were investigated.Results In 32 patients with knee-joint fixation method,27 were successful with median treatment duration 4.84 minutes.Meanwhile in other 179 ones with lower limbs fixation method,152 were successful with median duration 7.96 minutes.And the duration difference between two methods was found (P<0.05).Conclusion Knee-joint fixation may help significantly shorten the median treatment duration for the treatment of intussusception with air enema in children.

Background Radiofrequency thermocoagulation is one of the effective therapies for trigeminal neuralgia.Corneal nerve is important substance of radiofrequency thermocoagulation ocular surface,which support the normal structure and function of cornea.Most of corneal nerves come from ophthalmic branch of trigeminal nerve.However,the change of ocular surface microenvironment following radiofrequency thermocoagulation treatment in the patient with trigeminal neuralgiais unclear.Objective This study was to analyze ocular surface change after radiofrequency thermocoagulation therapy in patients with trigeminal neuralgia.Methods Twenty-eight eyes of 28patients with trigeminal neuralgia underwent radiofrequency thermocoagulation therapy were enrolled in this study.The contralateral eyes were regarded as the control group.The central corneal sensitivity,function of lacrimal secretion (Schiemer 1 test),tear break-up time(BUT),corneal fluorescence staining and laser scanning confocal microscopic examination were performed before and after surgery in operative eyes and compared with the fellow eye.Informed consent was obtained before any relevant medical procedure from each patient.Results No significant differences was found before surgery in the central corneal sensitivity,the Schiemer Ⅰ test,BUT,corneal fluorescence staining and densities value of corneal subepithelial nerve plexus between the treating eyes and fellow eyes(Z =-1.511,-1.119,-0.428,-0.378,-0.854; P =0.131,0.263,0.669,0.705,0.393).1n the third day after radiofrequencythermocoagulation therapy,compared with pre-treatment,no significant differences were seen in BUT result,Schirmer Ⅰ test and the score of ocular surface fluorescence staining (Z =-0.620,-0.315,-1.732;P =0.535,0.753,0.083).Corneal sensitivity and subbasal nerve density were lowed 3 days after surgery (Z =-2.708,-2.813 ; P =0.007,0.005).One month after treatment,differences of all indexes mentioned above showed statistical significance between treating eyes and fellow eyes(Z=-3.888,-2.373,-3.311,-2.535 ; P =0.000,0.018,0.001,0.011).The corneal subepithelial nerve was thinner and sparse and dendritic cells on the cornea were found in the eyes received radiofrequency thermocoagulation therapy.Conclusions The secretion of tears and the stability of tear film are poorer and the corneal sensitivity and subbasal nerve density are affected in a certain extent in the eye received radiofrequency thermocoagulation therapy.